This invention relates to a novel process for purifying and processing recombinant human papillomavirus virus-like particles (VLPs), which results in compositions suitable for vaccine use which have greater stability. Also, this invention relates to the VLPs made by this process.
Recombinant human papillomavirus (HPV) virus-like particles (VLPs), contain either L1 or a combination of L1 and L2 protein, but do not contain viral nucleic acids. They can be expressed in a variety of host cell types including yeast and insect cells and are attractive candidates for vaccine development to prevent genital HPV infection and the subsequent development of genital warts and/or cervical cancer. In animal studies, purified VLPs have been shown to induce high titers of antibodies against conformational type specific L1 epitopes. These antibodies neutralize homologous virions in in-vitro assays and protect against experimental challenge in several animal models.
xe2x80x9cMaturationxe2x80x9d, i.e., a change in stability, structural definition and other properties of VLPs have been observed with VLPs during purification, processing and storage. While not wishing to be bound by theory, it appears that this is due, at least in part to changes in intermolecular disulfide bond formation which is required for the assembly and further stabilization of virions.
It is important for a vaccine formulation to be stable. Thus, it would therefore be desirable to make stable VLPs which also maintain immunogenicity during storage.
It has been found, in accordance with this invention, that by subjecting papillomavirus L1 or L1+L2 protein to a maturation process, virus-like particles are produced which have improved antigenicity, size distribution, and stability. Thus this invention relates to a method for making human papilloma virus (HPV) virus-like particles (VLPs) comprising the steps of:
a) expressing HPV L1 or L1+L2 proteins;
b) at least partially purifying the proteins; and
c) subjecting the at least partially purified proteins to a maturation step.
There are various maturations processes encompassed by this invention, including incubation at an elevated temperature, glutathione facilitated thiol oxidation, exposure to a metal surface and exposure to light. One preferred maturation process is the step of incubating the at least partially purified proteins at an elevated temperature. Thus, a specific embodiment of this invention is a method for making HPV VLPs comprising the steps of:
a) expressing HPV L1 or L1+L2 proteins;
b) at least partially purifying the proteins; and
c) incubating the at least partially purified proteins at an elevated temperature.
In preferred embodiments of this invention, the proteins are recombinantly produced. Further, in other preferred embodiments, the elevated temperature is from about 30xc2x0 C. to about 45xc2x0 C. In a particularly preferred embodiment, the temperature is about 37xc2x0 C.
In another embodiment, the at least partially purified VLPs are treated with either glutathione or oxidized glutathion as a maturation step. The resulting matured VLPs are essentially the same as the heat-treated ones.
As the VLPs produced by this method can be differentiated from those produced without the maturation step, this invention also is directed to virus-like particles (VLPs) made by the process of expressing an L1 or L1+L2 proteins, at least partially purifying the proteins, and subjecting the at least partially purified proteins to a maturation step. This invention is also directed to vaccine compositions which contain the VLPs so produced.
Another aspect of this invention is a method of inducing an immune response in an individual comprising administering to the individual an effective amount of the vaccine composition comprising VLPs which were subjected to a maturation step.